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INTESTINAL Publication-derived

Patient-Derived Organoid (PDO) Culture and Drug Sensitivity Testing for Metastatic Colorectal Cancer

Source Ooft et al., 2019 · Netherlands Cancer Institute, Amsterdam · 10.1126/scitranslmed.aay2574

👤 Salo N. Ooft, Fleur Weeber, Krijn K. Dijkstra, Chelsea M. McLean, Sovann Kaing, Erik van Werkhoven, Luuk Schipper, Louisa Hoes, Daniel J. Vis, Joris van de Haar, Warner Prevoo, Petur Snaebjornsson, Daphne van der Velden, Michelle Klein, Myriam Chalabi, Henk Boot, Monique van Leerdam, Haiko J. Bloemendal, Laurens V. Beerepoot, Lodewyk Wessels, Edwin Cuppen, Hans Clevers, Emile E. Voest ⏱ 20 days 📋 6 phases 🧫 Patient-Derived Tumor Organoids (PDOs) from metastatic colorectal cancer lesions

Abstract

This protocol describes the establishment of patient-derived tumor organoids (PDOs) from metastatic colorectal cancer biopsies and their functional testing against irinotecan-based and combination chemotherapy regimens to predict clinical response. The method enables turnaround time of approximately 2-3 weeks and can identify non-responders to irinotecan-based therapies with >80% accuracy.

Cell source
Patient-Derived Tumor Organoids (PDOs) from metastatic colorectal cancer lesions
Application
Drug sensitivity prediction for chemotherapy response in metastatic colorectal cancer

Protocol overview

32 steps across 6 phases

Patient Biopsy Collection and Sample Processing Day 0–1
  1. 1 Collect tumor biopsy
  2. 2 Prepare biopsy collection medium
  3. 3 Dissociate biopsy tissue
  4. 4 Count and wash cells
PDO Culture Establishment and Expansion Day 0–10
  1. 1 Culture cells in CRC growth medium
  2. 2 Monitor organoid formation
  3. 3 First passage and cell expansion
  4. 4 Quality control: mycoplasma testing
  5. 5 Quality control: PDO authentication
PDO Dissociation and Preparation for Drug Screening Day 10–14 (before drug exposure)
  1. 1 Dissociate PDOs into single cells
  2. 2 Re-plate dissociated cells for organoid reformation
  3. 3 Harvest reformed organoids
  4. 4 Count organoids
  5. 5 Prepare organoid suspension for plating
Drug Screening Setup and Exposure Day 14–20 (6-day drug exposure)
  1. 1 Dispense organoids into 96-well plates
  2. 2 Overlay with CRC growth medium
  3. 3 Prepare drug matrices for irinotecan monotherapy
  4. 4 Prepare drug matrices for combination therapy (5-FU + Irinotecan)
  5. 5 Prepare drug matrices for combination therapy (5-FU + Oxaliplatin)
  6. 6 Establish positive and negative controls
  7. 7 Obtain baseline viability measurement
  8. 8 Incubate plates with drugs
Viability Assessment and Data Analysis Day 20 (end of 6-day drug exposure)
  1. 1 Measure day 6 viability
  2. 2 Calculate growth rate inhibition (GR) metrics
  3. 3 Fit dose-response curves (DRCs)
  4. 4 Identify concentration with largest window of effect
  5. 5 Calculate GR scores
  6. 6 Apply classification thresholds
  7. 7 Assess interobserver reproducibility
Genomic Characterization Concurrent with PDO establishment
  1. 1 Extract DNA from biopsied material
  2. 2 Perform targeted gene panel sequencing
  3. 3 Perform whole-genome sequencing (optional)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ooft et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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